The field of this invention is mammals comprising xenogeneic tissue, and in particular xenogeneic hepatocellular tissue.
The liver is a critically important organ for monitoring and adjusting plasma constituents. Hepatocytes are active in controlling levels of blood glucose, lipids and cholesterol, and a number of plasma proteins, including albumin, fibrinogen and prothrombin, and several complement factors. The structure of a liver lobule is that of a hexagon with portal triads at each corner, where each triad contains branches of the hepatic portal vein, hepatic artery and bile duct, so that each hepatocyte is in a close association with the vascular system.
Hepatocytes synthesize triglycerides, cholesterol and phospholipids. Much of the lipid synthesized is then packaged with proteins and released into the circulation as VLDLs, providing a source of fatty acids for all cells. Hepatocytes also synthesize the enzyme essential for formation of cholesterol esters in HDL, remove chylomicron fragments from the circulation, and are an indirect source of LDLs, which are formed in plasma from VLDLs depleted of fatty acids. Balancing the lipoprotein levels and cholesterol content in the circulation has proven to be a critical factor in vascular disease.
Glucose from the blood is stored by hepatocytes in the form of glycogen, which is a major source of glucose for other cells in the body. During meals with high glucose, insulin increases the ability of hepatocytes to synthesize glycogen. As blood glucose drops, glucagon and epinephrine increase the ability of hepatocytes to degrade glycogen. Enzyme deficiencies associated with glycogen deficiencies can result in storage diseases. The liver also has other specialized function other than glucose storage, including: detoxification; synthesis of critical plasma proteins, such as coagulation proteins, alpha-1 antitrypsin, and albumin; amino acid and ammonia metabolism; heme synthesis; and vitamin and cofactor biosynthesis.
Despite its specialized functions, the liver has a unique regenerative capacity. After partial hepatectomy, the liver mass is restored by division of fully differentiated hepatocytes. Even in adults, these cells have a tremendous replicative ability. The existence of liver stem cells remains controversial, but such cells may be active in liver growth after severe injury.
The response of hepatocytes to tissue damage is mediated by several cytokines. Immediately after an injury, hepatocytes undergo a priming phase in which they become competent to enter the cell cycle. This phase is characterized by expression of the proto-oncogenes c-myc and c-jun. The primed cells are then able to respond to cytokines such as epidermal growth factor (EGF), tumor growth factor (TGF-xcex1), Interleukin-6 (IL-6), and hepatocyte growth factor (HGF). TGF-xcex1 is synthesized by hepatocytes and acts as an autocrine factor. The in vivo response of hepatocytes to growth factors is discussed in references such as Y. Yamada et al., Am J Pathol. 152:1577-89 (1998); D. E. Cressman et al., Science 274:1379-83 (1996); R. Taub, FASEB J.10:413-27 (1996); N. Fausto et al., FASEB J. 9:1527-36 (1995); Webber et al., Hepatol 19:489-497 (1994).
Certain viruses such as hepatitis viruses show great specificity for infecting hepatocytes. Several hundred million people worldwide suffer from chronic hepatitis B virus (HBV) or hepatitis C virus infection which greatly increases their risk of developing liver cirrhosis and/or hepatocellular carcinoma (HCC). Medical therapy is generally not curative, and when available, transplanted livers can become re-infected. The only animals that can be infected with human hepatitis B virus (HBV) or human hepatitis C virus (HCV) are humans and chimpanzees, and the major tissue that is productively infected is the liver, although there have been reports of infected stromal cells.
Although in vitro models of hepatitis B and C have been used to study hepatitis virus infection (see e.g., Sureau, Arch. Virol.8:3-14 (1993); P. Lampertico et al., Hepatology 13:422-6 (1991); and N. Bishop et al., J Med Virol. 31:82-9 (1990), these models are limited as to the study of disease progression. Gene expression in the in vitro models is altered from normal in vivo expression in hepatocytes. Primary hepatocyte cultures are susceptible to infection for only a few days, if at all, and do not produce the characteristic infectious particles. Human hepatitis D virus (HDV) requires envelope proteins produced by HBV, and therefore can only infect cells susceptible to HBV. The need for a good experimental system having cells that are susceptible to productive infection by viruses such as the hepatitis viruses, and other hepatic pathogens, remains.
The field of medicine relies heavily on animal models. These models provide a means of analyzing the effect of viruses and other pathogens, cytokines, environmental factors, hormones, diet, and the like. Without animal models, it is extremely difficult to perform controlled experiments. An animal model having viable human tissue provides numerous advantages over other systems such as in vitro cultured tissue. One can investigate the effect of agents on the tissue at various stages in the development of the disease. The interactions of cells, secreted age tissue can also be analyzed. A xenogeneic animal model further provides a means of testing the effect of factors and other agents on cells that are difficult to maintain in culture. Short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time may be examined.
In view of the many important functions performed by the liver, it is of substantial interest to develop and provide animal models comprising functional human hepatocytes that remain viable for extended periods of time. An animal model would permit investigation of the function and dysfunction of hepatocytes, the etiology of disease and the effect of pathogens and therapeutic drugs.
Many different approaches for creating an animal model for liver disease using hepatocellular transplantation have been tried over the years. Hepatocytes of the same or similar species can be stably transplanted into the liver via the spleen or portal vasculature and shown to function in a hepatocyte specific manner. While hepatocellular transplantation within the same or related species has been established, see e.g., Rhim et al. Science 263:1149-1152 (1994), the creation of a mouse that can persistently harbor functional human hepatocytes and is susceptible to infection with HBV or HCV has not been demonstrated. Previous mouse or rat models show a low rate of persistence of hepatocyte function (K. Sanhadji et al., Bone Marrow Trans. 9:77-82 (1992); M. Fontaine et al., J. Ped. Surgery 30:56-60 (1995)). Transgenic mice expressing the hepatitis B genome replicates the virus, resulting in viremia, but not a normal course of hepatitis infection (M. J. Araki et al., Proc Natl Acad Sci USA 86:207-11(1989); M. B. Guidotti et al.,J. Virol 6:6158-69 (1995)). Chimpanzees and other higher primates remain the only species besides humans susceptible to infection with hepatitis B or C viruses.
There is thus a need in the art for animal models that allow the study of human liver dysfunction, e.g., dysfunction caused by pathogenic or parasitic infection or exposure to chemical agents. There is also a need in the art for a system that allows the study of normal human liver development and function.
Non-human mammalian hosts are provided, comprising functional human hepatocytes. Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host in conjunction with an agent, e.g., one or more activator that stimulates signaling through the human hepatocyte growth factor receptor (hHGFR). In one embodiment, the human hepatocytes are maintained in the host by administration of one or more agent that stimulates human hepatocyte growth factor receptor, either continuously (e.g., via an implanted catheter or intravenous apparatus) or in discrete, regular dosages of the agent (e.g., via intravenous injections or oral dosages). The human hepatocytes are able to survive and function in the host animal for a period of over 5 months. The chimeric animal has broad applicability in the study of human infectious diseases with hepatocellular tropism, degenerative and metabolic diseases of the human liver, and toxic or carcinogenic agents that target the human liver.
The invention also provides a method for enhancing the transplantation and/or maintenance of the human hepatocytes by the administration of growth factors, angiogenic factors, cytokines, or other agents that further promote the colonization and growth of the human hepatocytes in the mammalian host. In a specific embodiment, the invention provides enhancement of transplantation using a factor, e.g., FGF, that enhances vascularization at the transplantation site.
It is an object of the invention to provide an animal model for hepatitis infections, and particularly HBV, HDV and HCV infection.
It is another object of the invention to provide an animal model for human parasitic infection in which the parasite must pass through a liver phase, e.g., the human malaria parasites Plasmodium vivax and Plasmodium falciparum. 
It is yet another object of the invention to provide an animal model for human disorders involving exposure to chemicals or toxins, such as alcoholic cirrhosis.
It is yet another object of the invention to provide an animal model for studying normal human liver development and function.
It is yet another object of the invention to identify the efficacy of gene therapeutics to human liver cells, e.g. the transfer of genes with vectors specific to human and/or liver cells, or gene therapy to treat viral infections.
It is an advantage of the invention that the effect of agents on human hepatocytes are functional in vivo for an extended period of at least five months or more, and thus can be examined at various stages of development, pathogenic infection, or toxicity.
It is another advantage of the invention that the chimeric animals provide a means of testing the effect of factors and other agents on human hepatocytes, which are difficult to maintain in culture.
It is yet another advantage of the invention that the human hepatocytes in the chimeric animals are in an in vivo setting, and thus associate with other cells specific to an in vivo setting, e.g., short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time.
It is yet another advantage that the chimeric animals of the invention may be created in animals with various genetic backgrounds.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the chimeric animals as more fully described below.